Biological Conservation 184 (2015) 278–289
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Biological Conservation
journal homepage: www.elsevier.com/locate/biocon
Ghosts of glaciers and the disjunct distribution of a threatened California moth (Euproserpinus euterpe) ⇑ Daniel Rubinoff a, , Michael San Jose a, Paul Johnson b, Ralph Wells c, Ken Osborne d, Johannes J Le Roux e a 310 Gilmore Hall, Department of Plant and Environmental Protection Sciences, University of Hawaii, Honolulu, HI 96822, United States b Pinnacles National Park, 5000 Hwy 146, Paicines, CA 95043, United States c 303 Hoffman Street, Jackson, CA 95642, United States d Osborne Biological Consulting, 6675 Avenue Juan Diaz, Riverside, CA 92509, United States e Centre for Invasion Biology, Department of Botany and Zoology, Stellenbosch University, Matieland 7602, South Africa article info abstract
Article history: The Kern Primrose Sphinx moth (Euproserpinus euterpe) is a threatened moth twice thought to have gone Received 4 October 2014 extinct. It was historically known only from a small basin in the southern Sierra Nevada of California, but Received in revised form 12 January 2015 a new putative population was recently discovered 115 km to the west. Analysis of both nuclear and Accepted 13 January 2015 mitochondrial DNA suggest discontinuous genetic breaks between the Kern Primrose Sphinx, its closest Available online 21 February 2015 relative the Phaeton Sphinx (Euproserpinus phaeton), and at least one additional species discovered during the course of this study. Geographic distance is well correlated with genetic distance within species, but Keywords: not between species. Genetic discontinuities show the influence of past glacial events and suggest recent Lepidoptera range expansions, though not always congruent with other phylogeographic studies from the region. Our Sphingidae California phylogeographic results demonstrate that past glacial events, the altitudinal suppression of suitable habi- Phylogeography tat, and isolation may have been more important than population-level ecological factors such as differ- Endangered species ences in habitat (e.g. sand dunes, oak forest, montane grasslands) in promoting speciation. Effective conservation of the genetic diversity of the widespread Phaeton Sphinx and its two geographically restricted relatives requires genetic data at the population level because relatively few localized popula- tions harbor most of the genetic variation. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction The issue of monophyly, evolutionarily significant units (Moritz, 1994), and the controversy in the delimitation and conservation of The conservation of rare species and unique populations is usu- threatened species and populations has been intensively studied in ally guided by measures of genetic diversity, as this diversity often many North American mammals (Leonard et al., 2005; Ramey correlates with geographic and ecological patterns of isolation et al., 2006; Vignieri et al., 2006) and birds (e.g. Coulon et al., (Avise, 1998; Dimmick et al., 1999; Moritz, 1994, 2002). Conserva- 2008; Delaney and Wayne, 2005), but rarely in endangered insects tion of rare species whose genetic diversity is decoupled from geo- (e.g. Gompert et al., 2006; Saarinen et al., 2009), despite evidence graphic distance between populations poses an added challenge that insects are at least as threatened by extinction as other groups because discovery of populations of conservation importance (McKinney, 1999). requires additional scrutiny, not just finding new localities. The California Floristic Province is a global biodiversity hotspot Additionally, the causes of genetic discontinuity have broad rele- (Lapointe and Rissler, 2005), making it the focus of many phylogeo- vance to our understanding of microevolutionary processes such graphic studies, and of high conservation value. Most research has as speciation and its mechanics. When a species receives legislative focused on examining the importance of past vicariant events in protection, its evolutionary independence and, thus, justification interpreting congruence, or the lack thereof, in patterns of genetic for protected status can be called into question (e.g. Ramey et al., discontinuity in modern populations across many taxa (Calsbeek 2006; Vignieri et al., 2006). This can be particularly controversial et al., 2003; Lapointe and Rissler, 2005), including salamanders if the protected species is only moderately diverged from adjacent, (Jockusch and Wake, 2002), newts (Kuchta and Tan, 2005, 2006), non-threatened sister taxa. frogs (Macey et al., 2001), woodrats (Matocq, 2002), titmice (Cicero, 1996; Lapointe and Rissler, 2005), snakes and lizards (Feldman and Spicer, 2006), beetles (Caterino and Chatzimanolis, ⇑ Corresponding author. http://dx.doi.org/10.1016/j.biocon.2015.01.023 0006-3207/Ó 2015 Elsevier Ltd. All rights reserved. D. Rubinoff et al. / Biological Conservation 184 (2015) 278–289 279
2009; Polihronakis and Caterino, 2010a; Polihronakis and Caterino, and concerns of over-collecting and the ecological impact of an 2010b; Polihronakis and Caterino, 2012; Polihronakis et al. 2010), exotic weed, Erodium cicutarium (L.) L’Hér. ex Aiton Geraniaceae, spiders (Hedin and Carlson 2011; Hedin et al., 2013), and walking E. euterpe became the first moth species to receive federal protec- stick insects (Law and Crespi, 2002). tion in the United States (USFWS, 1980). Shortly after being listed Overwhelmingly, these studies have focused on taxa that rely as Threatened, researchers failed to find the moth for several years on relatively mesic habitats, which expand and connect during gla- and it was again feared extinct until its rediscovery in the Walker cial periods and contract into isolation during drier, warmer peri- Basin in the 1990s (Osborne, 1999; Shields, 1990) and has persist- ods (e.g. Calsbeek et al., 2003; Lapointe and Rissler, 2005; ed since (Osborne, pers obs). In 2006, an additional population of E. Schoville et al., 2011). The moth genus Euproserpinus (Sphingidae) euterpe was discovered at Carrizo Plain National Monument, provides an opportunity to examine the opposite, because a desert 115 km west of the Walker Basin population (Jump et al., 2006). species will become isolated when mesic conditions spread (Fig. 1). Whereas E. euterpe, appears to be restricted to the Walker Basin The genus is also of high conservation relevance because E. euterpe and the vicinity of Carrizo Plain National Monument, the Phaeton (Edwards, 1888), the Kern Primrose Sphinx moth, is federally listed Sphinx (E. phaeton) occurs allopatrically from E. euterpe, 27 km east as a threatened species, and resources are available to protect it of Walker Basin in Kelso Valley and 53 km away at Walker Pass, and change land management regimes given data on its distribu- with widely-scattered but localized populations distributed north- tion and species status. east to Lassen county and south across the Mojave and Colorado Because many previous phylogeographic studies have focused Deserts (Fig. 2). While there are some morphological differences on Central and Southern California as important areas for under- in larval and adult maculation between the two species (Jump standing the impacts of glaciation and geology in shaping genetic et al., 2006), their close proximity at Walker Basin and nearby discontinuity (e.g. Alexander and Burns, 2006; Chazimanolis and localities suggests the possibility for gene flow. For E. euterpe to Caterino, 2007; Feldman and Spicer, 2006; Kuchta 2007; Kuchta be restricted to two disjunct populations 115 km apart, and yet and Tan, 2006; Law and Crespi, 2002; Macey et al., 2001; to be reproductively isolated from E. phaeton just 27 km away, sug- Matocq, 2002; Rich et al., 2008; Rodríguez-Robles et al., 2001; gests one of two scenarios. Either a taxonomic error should be cor- Rubinoff and Sperling, 2004; Sandoval et al. 1998; Sgariglia and rected by lumping a federally listed threatened species with a Burns, 2003; Starrett and Hedin, 2007), Euproserpinus appears to widespread congener, or else the current taxonomy is valid, being be an ideal study subject for improving our understanding of this the result of a less parsimonious and remarkably incongruous pat- phenomenon. Most urgently, because Euproserpinus contains a fed- tern of speciation and biogeographic isolation. erally listed species, E. euterpe, understanding the genetic structure Specifically, we seek to address the following questions: Are E. of the genus and its underlying determinants across California will euterpe and E. phaeton genetically isolated? Is the newly discovered be important in identifying the threatened species, managing high- Carrizo Plain population in fact the threatened E. euterpe, as sug- ly restricted populations, and prioritizing areas for conservation. gested by morphological characters as presented by Jump et al. Recent work has confirmed that insects represent the majority of (2006)?IfE. euterpe is truly restricted to just one or two disjunct California Floristic Province’s endemic biodiversity but are rarely locations, what are the patterns of phylogeographic relationships considered in conservation planning, perhaps due to a lack of even among the populations and how do they contrast with E. phaeton, basic faunistic or genetic data (Caterino, 2007). Even with legisla- which occurs across thousands of km2 of desert? What are the tive protection, the genetic status of E. euterpe has remained broader biogeographic and ecological implications of the genetic unstudied and the phylogeography of the genus completely relationships across the genus? And finally, how does the phylo- unknown; essential components to circumscribing species, and geography of Euproserpinus elucidate both applied and theoretical justifying and effectively directing resources toward those that conservation planning on a regional scale? are endangered. The current default hypothesis that has been used to justify Euproserpinus euterpe was thought to be restricted to the Walk- conservation action, is that the E. euterpe populations, which occur er Basin, Kern Co. California, an area of only 15 km2 isolated in the in the Walker Basin and Carrizo Plain, are more closely related to southern Sierra Nevada (Tuskes and Emmel, 1981). Owing to its each other than either is to the many E. phaeton populations sur- limited distribution, habitat loss due to farming and overgrazing, rounding them. If this is true, then E. euterpe populations will clus- ter under population level analyses, with all E. phaeton populations, even those geographically close to E. euterpe, being more genetically distant, and monophyletic. If the E. euterpe populations do not cluster together, then geographic distance pre- dicts genetic proximity, the two E. euterpe populations will not be each other’s closest relatives, and the validity of the species and its listed status would be called into question. Because we are interested in understanding the probability that different populations may represent distinct species, including newly discovered ones in Carrizo Plain and the Central Coast area, we began with the most conservative approach by assuming that taxa are not distinct at the population level. In this way, the null hypothesis of genetic continuity is tested, rather than presumed.
2. Materials and methods
2.1. Study species, specimen collection, and DNA extraction Fig. 1. Hypothetical change in habitat distribution for Euproserpinus during (A) interglacial and (B) glacial periods. Although the mountains separating Euproser- The three described species of Euproserpinus (E. phaeton, E. pinus were never glaciated, habitat would have been unsuitable for the moths. Agriculture and habitat alteration in the Central Valley appears to have isolated E. wiesti, and E. euterpe) are small diurnal hawkmoths highly adapted euterpe.in two remaining populations. to desert climates. Their pupae may stay underground for years, 280 D. Rubinoff et al. / Biological Conservation 184 (2015) 278–289
Fig. 2. (A) Map of California indicating the location of study areas with circles representing different Euproserpinus populations. (B) Heat map of pairwise Ust (lower diagonal) and Jost’s D values (upper diagonal) of Euproserpinus populations. Darker colors indicate higher genetic differentiation; lighter colors indicate lower genetic differentiation. awaiting a winter of adequate rainfall. We attempted to collect genomic DNA was extracted using the Qiagen DNeasy extraction samples from throughout the entire known distributions of E. kit (Qiagen, USA) from small pieces of abdomen or individual legs. phaeton and E. euterpe, plus E. wiesti from a single locality. Adults Voucher specimens are held in the University of Hawaii’s Insect emerge and fly for perhaps two weeks in late winter or very early Museum’s cryogenic collection and by K.O., P.J, and the Pinnacles spring when few other insects are active, nighttime temperatures National Park Museum collection. can fall below 0 °C, and their annual Onagraceous host plants have just begun to grow in the short-lived spring. During the frequent 2.2. PCR amplification and DNA sequencing droughts in these xeric regions, the moths may hold over for years as pupae, not emerging at all and giving the impression that a The mitochondrial gene cytochrome oxidase c subunit 1 (COI) population has disappeared. Further, because desert precipitation was amplified in two parts for all specimens using the universal and temperatures are highly variable, distant populations may be primers LCO-1490 and HCO-2198 and Jerry-k485 and Pat-k508 genetically isolated because of temporal isolation due to consistent (Folmer et al., 1994; Simon et al., 1994). Each 50 lL PCR reaction differences in adult emergence during the course of a season. Thus, contained ca. 50 ng of genomic DNA, 200 lM of each dNTP (Bioline, for this study, locating and collecting samples of the populations USA), 25 pmol of each primer, 5 U Taq DNA polymerase (Bioline, across the deserts of California and Arizona has taken more than USA), 1 Â PCR reaction buffer, 1.5 mM MgCl2. PCR cycles consisted a decade (from 1997 to 2013), led to the discovery of many new of initial denaturation of 95 °C for 5 min; 35 cycles for denaturation localities, and may still be incomplete. at 94 °C for 30 s, annealing at 53 °C for 60 s, elongation at 72 °C for During the study, one of the authors (P. Johnson) discovered a 90 s; and final extension at 72 °C for 10 min. Due to unexpectedly new population of Euproserpinus at Pinnacles National Park, San deep divergences between some clades for the COI data (see Benito Co., the farthest northwest the genus has been found. He results) we also sequenced a second, slower evolving nuclear gene, subsequently discovered more populations in San Luis Obispo elongation factor 1 – alpha (EF1a), for representative taxa from and Monterey counties. These moths appear darker in color than each clade. EF1a was amplified using the primers Oscar and Bosie any other Euproserpinus taxa and inhabit open sandy areas adja- (Haines and Rubinoff, 2012) and PCR cycles consisted of initial cent to riparian habitats, oak savannah and openings in chaparral, denaturation at 95 °C for 5 min followed by 35 cycles at: 94 °C remarkably mesic locations for a genus typical of scrub and barren for 30 s, 55 °C for 60 s, and 72 °C for 90 s; and 72 °C for 10 min. desert. All PCR amplifications were done in an MJ PTC-200cycler (MJ In addition, we collected specimens of E. wiesti, the only other Research, USA). Amplified DNA fragments were purified using the member of Euproserpinus, for use as an outgroup taxon in phyloge- QIAquick PCR Purification Kit (Qiagen, USA) and sequenced in both netic analyses. This species had been considered for federal protec- directions using the ABI PRISM BigDye Terminator Cycle Sequenc- tion and is restricted to sand dune habitats along the Front Range ing Ready Reaction kit and an automated ABI PRISM 377XL DNA of the Rocky Mountains. Locality data can be found in Table 1. Total sequencer (Applied Biosystems, USA). D. Rubinoff et al. / Biological Conservation 184 (2015) 278–289 281
Table 1 Collection and genetic information for Euproserpinus from 30 collection sites. Each site is coded to correspond to population designation for population genetic analysis, number of DNA sequences generated (N), and haplotypes observed at the site.
Species Population Locality N Haplotypes E. euterpe Carrizo Plain CA: San Luis Obispo Co., Carrizo Plain 15 H04, H08, H56, H63, H64 E. euterpe Carrizo Plain CA: Santa Barbara Co., Cuyama Valley 2 H04 E. phaeton Imperial Co. CA: Imperial Co., Imperial Sand Dunes 6 H01, H18, H23, H57 E. phaeton Lassen Co. CA: Lassen Co., Doyle 6 H12, H27, H28, H35, H58 E. phaeton Lassen Co. CA: Lassen Co., Ft. Sage Mtn. 6 H12, H20, H28, H39 E. nsp Monterey Co. CA: Monterey Co., Stockdale Mtn. 7 H03, H38 E. nsp Monterey Co. CA: Monterey Co., Vineyard Cyn Rd. 3 H03 E. weisti New Mexico NM: Quay Co. 6 H09, H48, H49 E. nsp Pinnacles National Park CA: San Benito Co., Pinnacles National Park 4 H05, H41 E. phaeton Riverside Co. CA: Riverside Co., Anza Valley 45 H01, H02, H07, H11, H13, H26, H31, H53, H54, H55 E. phaeton Riverside Co. CA: Riverside Co., Black Hills 2 H02, H52 E. phaeton Riverside Co. CA: Riverside Co., Gavilan Hills 1 H02 E. phaeton Riverside Co. CA: Riverside Co., Temecula Wash 12 H01, H06, H11, H32, H33, H34 E. phaeton Riverside Co. CA: Riverside Co., Wilson Valley 3 H02, H14 E. phaeton San Diego Co. CA: San Diego Co., Campo 4 H01, H19 E. phaeton San Diego Co. CA: San Diego Co., Jacumba 1 H01 E. phaeton San Diego Co. CA: San Diego Co., La Posta Road 23 H01, H07, H18, H21, H22, H06, H65, H68, H69, H70, H71, H72, H73 E. phaeton San Diego Co. CA: San Diego Co., Mason Valley 2 H61, H62 E. phaeton San Diego Co. CA: San Diego Co., Ranchita 9 H02, H40, H42, H43, H44, H45, H46, H47 E. phaeton San Diego Co. CA: San Diego Co., Things Valley 8 H01, H21, H22, H74, H75 E. nsp Shell Creek CA: San Luis Obispo Co., Shell Creek 12 H03, H05, H24, H25 E. phaeton Ventura Co. CA: Ventura Co., Lockwood Cr. Rd. 4 H01, H36, H37 E. phaeton Ventura Co. CA: Ventura Co., Lockwood Valley 6 H01, H16 E. euterpe Walker Basin CA: Kern Co., Walker Basin 9 H10, H29, H30, H60 E. phaeton Walker Pass CA: Kern Co., Highway 178 2 H02 E. phaeton Walker Pass CA: Kern Co., Kelso Valley 6 H02, H15 E. phaeton Walker Pass CA: Kern Co., Walker Pass 26 H01, H02, H06, H59, H66, H67 E. phaeton Walker Pass CA: Kern Co., Weldon 5 H01, H06 E. phaeton Yuma Co. AZ: Yuma Co., Yuma 6 H01, H15, H17, H76 E. phaeton Yuma Co. AZ: Yuma Co., Highway 80 4 H02, H50, H51
2.3. DNA sequence alignment We dated the branching events of the different populations of Euproserpinus by first determining if the COI dataset had an equal Sequence contigs were constructed, edited and aligned using rate of evolution throughout the tree (i.e. assumptions of a molecu- BioEdit version 7.0.5.3 (Hall, 1999) or Geneious 6.0.5 (Biomatters). lar clock) using MEGA 5.2.2 (Tamura et al., 2011). Dating analyses All unique sequences were deposited in GenBank (http://www. were conducted using BEAST 1.7.5 (Drummond et al., 2012). Our ncbi.nlm.nih.gov, Supplementary Materials Table 1). analysis followed Brower’s (1994) approximation of mtDNA pair- wise sequence divergence of 2.3% divergence per million years for Lepidoptera, corresponding to 0.0115 substitutions per site on 2.4. Phylogenetics and molecular dating the COI dataset. Because no fossils exist for Euproserpinus and there are no clear biogeographic calibration points to date nodes within Nuclear and mitochondrial datasets were tested separately for our tree, we used a strict molecular clock with a Yule process prior an appropriate nucleotide substitution model using jModelTest for model of speciation and ran the analysis for 1 108 gen- v2.1.3 under the Akaike information criterion with correction  erations. To guarantee that the MCMC chain had run long enough (AICc) (Darriba et al., 2012). This approach identified the HKY + C to get a valid approximations of the parameters, individual log files and HKY as the most appropriate models of sequence evolution were analyzed with Tracer v1.5 (Rambaut and Drummond, 2009) for the COI and EF1a genes respectively (Supplementary Materials to assess convergence and confirm that the combined effective Table 2). Each gene was analyzed separately and then concatenat- sample sizes for all parameters were larger than 200. All resulting ed using GARLI 2.0 (Zwickl, 2006) for Maximum Likelihood (ML) trees were combined using LogCombiner v1.5.3 (Drummond et al., and MrBayes 3.2.1 (Ronquist et al., 2012) for Bayesian inference 2012), with a burn-in of 25%. A single maximum clade credibility analyses. For all analyses, four independent Bayesian markov chain tree was then drawn using TreeAnnotator v1.7.5 (Drummond monte carlo (MCMC) runs were conducted in MrBayes, each with et al., 2012) and visualized using Figtree v1.4 (Rambaut, 2012). one hot chain and three cold chains. Each run started with a ran- We realize that our approach using relative rates for divergence dom tree, sampling every one thousand generations for 10 million estimation is imperfect (Britten, 1986; Lepage et al., 2007; Roger generations with a relative burn-in of 25% using default priors and Hug, 2006). Although it should not be regarded as more than except that the parameters statefreq, revmat, shape, and pinvar a broad estimate of divergence time, it provides a rough estimate were unlinked between partitions. We used Tracer 1.5 (Rambaut that may be compared with known climatic variation to inform and Drummond, 2009) to determine MCMC convergence for all plausible patterns of diversification. Bayesian analysis. For the GARLI analysis, we conducted ten ML tree searches with default settings using a random starting tree to find the tree with the best likelihood score. To assess branch 2.5. Population genetics and network analysis support one thousand Maximum Likelihood bootstrap replicates were conducted in GARLI, Maximum likelihood bootstrap trees The full COI dataset was used to reconstruct networks using sta- were summarized in Sumtrees v3.1.0 (Sukumaran and Holder, tistical parsimony (Templeton et al., 1992) as implemented in TCS 2010) with a minimum clade frequency of 50% and branch support version 1.21 (Clement et al., 2000). We decided to analyse the data was mapped onto the best scoring ML tree. Trees were visualized using a network in addition to traditional phylogenetic tree build- using FigTree v1.4.0 (Rambaut, 2012) and rooted with E. wiesti. ing approaches, not only because of its higher resolution, but also 282 D. Rubinoff et al. / Biological Conservation 184 (2015) 278–289 because networks are better suited, though not perfect, for infer- ring relationships at the intra-specific level, as these are often not hierarchical as assumed by traditional tree building methods (Posada and Crandall, 2001). Genetic variability of COI sequences was calculated using DNAsp (Librado and Rozas, 2009). Genetic diversity indices calculated included the number of haplotypes (Nh), haplotype diversity (h), nucleotide diveristy (p) and the num- ber of segregating sites (S). Tests of gene neutrality were also cal- 0.061237 0.90839 0.478710.61237 1.26963 1.19483 À culated using DNAsp for the different species/populations À À included: Fu and Li’s F⁄, Fu and Li’s D⁄, Tajima’s D and Fu’s F (Table 2)(Tajima, 1989; Fu and Li, 1993). Estimates of population genetic structure were calculated as 0.339 0.172 0.497 1.11173 1.34668 1.24341 À À À À analysis of molecular variance (AMOVA) and as population pair- Monterey Co. Pinnacles NM Shell Creek wise Ust values in Arlequin version 3.5 (Excoffier and Lischer, 2010). Population pairwise Jost’s D (Jost, 2008) values were also 0.878 0.34181 calculated in R (R Core Development 2013) using the R packages 0.34091 0.27344 À À À À E. nsp seqinR (Charif and Lobry, 2007) and APE (Paradis et al., 2004) using an R script from Pennings et al. (2011). A permutation test with 1000 replicates was conducted in R to test significance of Jost D values. Pairwise Ust estimates were used in subsequent Mantel 0.55 À tests and regressed against log-transformed geographical pairwise distances to test for isolation by distance using Arlequin 3.5 (Excoffier and Lischer, 2010). These results were visualized using 0.286 0.91004 0.47721 the R statistical environment (R Core Development, 2013). 0.747550.59727 0.34159 0.23149 À À À À Walker Basin Carrizo Plain
3. Results 0.907 À E. euterpe 3.1. DNA sequence alignment 2.41 1.1165 1.01239 1.27406 1.01621 1.13824 0.812113 À À For the full COI dataset, aligned DNA sequences were 1435 bp À À long. For the combined COI and EF1a sequence data the aligned sequences were 2197 bp long, requiring no gaps. All unique DNA sequences were deposited in GenBank (accession numbers 2.056 0.96861 1.72582 1.70249 À À KP263128-KP263236). À À 1.466 1.03446 3.2. Phylogenetic analysis and molecular dating 0.96179 0.8049 À À À À
The ML tree for the combined EF1a and COI data (Fig. 3)
revealed that E. euterpe from the Walker Basin formed a well- = 245, 1435 bp). n supported (78 BS, 1.0 PP) clade with Euproserpinus populations col- ( 26.888 2.14453 3.26492 3.07363 À À lected across the Central Valley in the vicinity of the Carrizo plain. À À In contrast E. phaeton from Walker Pass formed a clade with populations from Lassen Co. in the far north, and all the way south along the eastern flanks of the Sierra Nevada ranges through the Euproserpinus 8.344 1.51513 2.46639 2.38133 À À Mojave and Colorado Deserts to at least as far south as the Arizona À À and California borders with Mexico, and west to the Pacific Ocean. Samples from San Benito Co. (Pinnacles National Park), San Luis 0.768
Obispo Co. (Shell Creek) and Monterey Co. formed a distant and À basal (97 BS, 1.0 PP) clade to all other Euproserpinus taxa. The COI ML tree (Fig. 4) suggested that E. phaeton is paraphylet- ic, E. euterpe is closest to samples from Lassen Co. to the north and 0.024 0.88407 0.53551 1.042360.99519 0.69599 0.63967 À À À À Imperial Co. to the south. Samples from San Benito Co., San Luis Imperial Co Lassen Co Riverside Co San Diego Co Ventura Co Walker Pass Yuma AZ Obispo Co. and Monterey Co. are basal with respect to all Euproser- pinus taxa. The EF1a tree (Fig. 5) showed poor resolution. The lack 74.9 2.35813 5.00445 5.06986 À À of resolution for both the COI and EF1a trees indicates relatively E. phaeton À À ) 58 7 10 17 26 3 7 9 9 5 4 6 1 1 4 recent divergence between E. euterpe and E. phaeton, or repeated S ) 0.00163 0.00181 0.00261 0.00125 0.00151 0.00053 0.00075 0.00184 0.00216 0.00101 0.00095 0.00097 0.00014 0.00035 0.00116 sequences within species and populations of ) 0.808 0.86667 0.90909 0.74194 0.89362 0.64444 0.5857 0.93333 0.834 0.77778 0.66176 0.705 0.2 0.5 0.77273 )584 7 1626 4 7 7 9 4 5 6 2 2 4 l periods of genetic exchange through secondary contact and intro- h -value < 0.05. Nh COI gression after initial divergence. p Tests for molecular clock assumptions did not reject the null ⁄ hypothesis of equal evolutionary rates throughout the tree ⁄ (P = 0.95). Our dating analysis of the COI data based on relative rates (Brower, 1994) estimated the MRCA for Euproserpinus within California to 0.67 MYA (range 0.94–0.45 MYA, Fig. 6). The newly Fu’s F Fu and Li’s D Tajima’s D No. of Segregating sites ( Fu and Li’s F Sample sizeNo. of haplotypes ( 187 6 12 63 47 10 39 10 26 9 17 26 10 4 12 Haplotype diversity ( Nucleotide diversity ( Table 2 Bold values indicate discovered species diverged from the rest of Euproserpinus 0.3977 Genetic variability of D. Rubinoff et al. / Biological Conservation 184 (2015) 278–289 283
Fig. 3. Maximum likelihood tree for the concatenated dataset (COI, EF-1a). Support values above branches are Maximum Likelihood Bootstrap values/Bayesian Posterior Probabilities. Scale bar indicates the number of substitutions per site. Each branch tip represents multiple individuals.
MYA (range 0.56–0.25 MYA). E. euterpe diverged from E. phaeton population-level analyses such as AMOVA, Ust and Jost’s D values, 0.1543 MYA (range 0.24–0.07 MYA). more appropriate for recently diverged taxa, indicate that both populations of E. euterpe (Walker Basin and Carrizo Plain, Table 4) are significantly different from the other populations of Euproserpi- 3.3. Population genetics and network analysis nus in California. Various indices were calculated to explore within population genetic variability and test the neutrality of mutations in Euproser- 4.2. Population genetics pinus (Table 2). Analysis of molecular variance (AMOVA) indicates higher genetic variation between species but lower variance Our population level analysis not only confirms the genetic between populations within species (Table 3). Pairwise Ust and uniqueness of E. euterpe from Walker Basin, but also suggests that Jost’s D values between E. euterpe and E. phaeton populations the populations in the vicinity of Carrizo Plain represent additional (Table 4, Fig. 2) are congruent, with higher values in intraspecific lineages of this species, once thought to have a very restricted dis- population pairs than interspecific pairs. Due to the low number tribution; a remarkable result considering the proximity of the of populations (n < 3 for E. euterpe and E. nsp.) we were only able Walker Basin Euproserpinus to those of the Walker Pass E. phaeton. to conduct the Mantel test with E. phaeton (Fig. 7); it indicated a Yet these Walker Pass populations are in relatively close genetic significant correlation between genetic differentiation and geo- contact with E. phaeton that occur hundreds of kilometers to the graphic distance for this species. Network analysis indicated sig- north in the colder Great Basin sand dune habitats of Lassen Co. nificant intraspecific genetic structure, suggesting some isolation (where the moth flies in mid-April), and southern populations from between populations, but much greater isolation between each of the extreme deserts of Yuma, Arizona (where the moth flies in the species (Fig. 8). December and January). In short, there is more genetic diversity in the E. euterpe on opposite sides of the San Joaquin Valley than there is in E. phaeton between far northern California and the Mex- 4. Discussion ican border. The most divergent Euproserpinus in California is the newly dis- 4.1. Phylogenetics and molecular dating covered species from San Benito, Monterey, and northern San Luis Obispo counties. These appear to have been long-isolated from In both the combined analysis and the COI phylogeny (Figs. 3 other Euproserpinus lineages, despite now occurring within 60 km and 4), E. euterpe is placed within E. phaeton rendering E. phaeton of E. euterpe in San Luis Obispo Co. Our dated phylogeny indicates paraphyletic. These are recently diverged species (150 KYA) and that this taxon diverged from the rest of Euproserpinus around 400 due to incomplete lineage sorting, our phylogenetic analysis was KYA. Our age estimates should be regarded as approximate, but not able to differentiate between the two species. In contrast, broadly place the divergences of Euproserpinus in the Ionian Stage 284 D. Rubinoff et al. / Biological Conservation 184 (2015) 278–289
Fig. 4. Maximum likelihood tree based on the mitochondrial COI dataset. Support values above branches are Maximum Likelihood Bootstrap values/Bayesian Posterior Probabilities. Scale bar indicates the number of substitutions per site.
of the Pleistocene, in which there were cycles of glaciation and (Althoff et al., 2006; Rich et al., 2008). While the impacts of glacial warmer interglacial periods, the former of which might have iso- events on different organisms have varied in their nature, severity, lated populations of Euproserpinus (Fig. 1). While other recent phy- and spatial extent, genetic isolation caused by glacial cycles logeographic studies of insects over the same region found appears to be widespread across vertebrates, insects and spiders evidence of genetic isolation and population structure in the southern Sierra (Alexander and Burns, 2006; Feldman and (Polihronakis and Caterino, 2010a, b; Polihronakis et al., 2010), Spicer, 2006; Kuchta and Tan, 2006; Kuchta, 2007; Law and none of these revealed patterns that were even roughly congruent Crespi, 2002; Macey et al., 2001; Matocq, 2002; Rich et al., 2008; with Euproserpinus, possibly because these other taxa are montane/ Rodríguez-Robles et al., 2001; Sandoval et al., 1998; Sgariglia and mesic specialists. Perhaps the most dramatic contrast between the Burns, 2003; Starrett and Hedin, 2007). Because organisms differ phylogeography of Euproserpinus and other taxa from the region is in their habitat requirements and generation times, we would the genetic discontinuity typically seen separating east from west not necessarily expect the estimates of isolation events to be con- across the Transverse Ranges (e.g. Calsbeek et al., 2003; gruent across taxa; glacial maxima would have been periods of Chatzimanolis and Caterino, 2007; Caterino and Chatzimanolis, expansion for cool, mesic dependent species, and retraction for 2009) that is absent in Euproserpinus. Our data showed E. phaeton desert taxa such as Euproserpinus. in genetic contact around the Transverse Ranges, while E. euterpe Cold, wet periods greatly restricted desert and scrub habitat occurs to the north in the vicinity of the Carrizo Plain. during the Pliocene and Pleistocene (Axelrod, 1980). Glaciers would not have directly impacted Euproserpinus, because the areas 4.3. Phylogeography where they occur were apparently never glaciated, but climatic shifts would have pushed populations to lower elevations where Euproserpinus consistently demonstrates inconsistencies drier, warmer, suitable habitat remained. Populations may have between geography and genetic diversification (Figs. 1 and 8), with ended up in isolated desert refugia, divided by cooler and more E. phaeton being genetically connected over long distances across mesic biomes at higher elevations. The Walker Pass area would much of California, yet oddly isolated from nearby populations of have sustained a wetter and cooler climate, probably unsuitable E. euterpe in Kern and Santa Barbara counties. We suspect that for Euproserpinus (Axelrod, 1980). Our molecular dating estimates the reasons for the currently incongruous patterns of genetic isola- suggest that divergence between E. euterpe and E. phaeton coincid- tion are the combined artifacts of past climatic and more recent ed with the glacial periods about 250,000 years ago. While the gla- human activity, as discussed below. ciers are long gone, they appear to have had lasting impacts on Genetic isolation of populations in the southern Sierra Nevada Euproserpinus, promulgating a speciation event across the Southern has been shown across many taxa (Feldman and Spicer, 2006; Sierra. At some point in the glacial cycles, after the glaciers retreat- Kuchta, 2007; Kuchta and Tan, 2005), including other moths ed, secondary expansion back to higher elevations would have D. Rubinoff et al. / Biological Conservation 184 (2015) 278–289 285
Fig. 5. Maximum likelihood tree based on the mitochondrial EF-1a dataset. Support values above branches are Maximum Likelihood Bootstrap values/Bayesian Posterior Probabilities. Scale bar indicates the number of substitutions per site. Low variability in this gene reduced resolution and branch support. resulted in the proximal geographic, and remarkably disparate described in a future publication. In contrast to the geologically- genetic pattern we see between E. euterpe and the Walker Pass E. mediated events of the more distant past, the unexpected connec- phaeton today. Interestingly, most other phylogeographic studies tivity of E. euterpe in Walker Basin and Carrizo Plain may be due to in the region find an east/west division (e.g. Polihronakis and recent changes to a historic range that was once continuous across Caterino, 2010b), rather than the north/south split evident in the San Joaquin Valley. While it is now highly modified agricultural Euproserpinus. Again, we suspect this is because montane/mesic- land or degraded scrub, the southern valley was previously a dependent taxa have been the focus of most previous studies. patchwork of riparian and desert landscapes connecting the Walk- The relatively deeper divergence evident in the new species of er Basin with the Carrizo Plain. Thus, the current isolation of E. Euproserpinus from the Central Coast suggests an older origin. In euterpe into two contemporary refugia of suitable habitat might the past 2–5 million years two seaways may have existed, isolating be the reflection of a few hundred years of anthropogenic activity the Coast Ranges at the Monterey peninsula to the north and the on what was likely a much more widespread taxon across the San southern tip of the Coast Ranges to the south, (Hall, 2002; Yanev, Joaquin Valley. 1980). While the timing of the strong phylogeographic break in We presume that the genetic discontinuity between E. euterpe San Luis Obispo Co. is more recent than the hypothesized seaways in the Walker Basin and Carrizo Plain is due to the relatively recent that made the region an island during the Miocene, this appears to extinction of genetically and geographically intermediate popula- be one of the most significant frontiers for Euproserpinus, and at tions across the Southern San Joaquin valley. Alternately, it may least as important as the southern Sierra. The reasons for the break be that the genetic divergence between E. euterpe in the Walker in this region remain unclear, since all three Euproserpinus species Basin and Carrizo Plain is a robust indicator of long isolated use very similar habitats. Because the molecular clock estimates populations because the Sierra Nevada and Transverse ranges are suggest that this speciation event was only 400,000 years ago, it isolating factors for many species of mammals, reptiles and is possible that Euproserpinus was isolated on the Central Coast amphibians (Jockusch and Wake, 2002; Kuchta and Tan, 2005, islands and secondary, more recent, introgression is obscuring 2006; Leonard et al., 2005; Macey et al., 2001; Ramey et al., what used to be a deeper genetic division between the groups or, 2006; Vignieri et al., 2006). However, Euproserpinus is xerophilic, since the methods used to date our phylogeny are not robust, the and the pre-agriculture southern San Joaquin valley is unlikely to actual divergence may have occurred much later. have been much different from the current desert of the Carrizo The older origin, and genetic distinctiveness of the Central Coast Plain. Thus, Euproserpinus provides a contrasting example to the species supports its designation as a new species and it will be phylogeography of the mesophilic taxa previously studied in this 286 D. Rubinoff et al. / Biological Conservation 184 (2015) 278–289
Fig. 6. Bayesian chronogram of the COI dataset of Euproserpinus showing 95% confidence intervals of posterior dates as calculated in BEAST.
Table 3 Analysis of molecular variance.
Source of variation DF Sum of Variance Percent Fixation squares component variation indices
Among species 2 393.44 4.35 Va 77.42 FSC = 0.32 P- value = 0.00
Among populations 9 77.62 0.40 Vb 7.24 FST = 0.84 within species P- value = 0.00
Within populations 227 195.95 0.86 Vc FCT = 0.77 P- value = 0.00 Total 238 667.02 5.62 Fig. 7. The relationship between Ust and log of physical distances between Euproserpinus phaeton populations. Significance test based on 1023 permutations populations are same as haplotype network.
Table 4 Pairwise Ust values (below diagonal) and Jost’s D values (above diagonal) based on COI sequences (n = 245, 1435 bp).
Species Locality E. phaeton E. nsp E. euterpe Imperial Lassen Riverside San Ventura Walker Yuma Monterey Pinnacles Shell Carrizo Walker Co. Co. Co. Diego Co. Co. Pass Co. Co. NP Creek Plain Basin E. phaeton Imperial Co. 0 1.00000⁄ 0.26248 0.07449 0.19588 0.27969 0.32468 1.00000⁄ 1.00000⁄ 1.00000⁄ 1.00000 1.00000⁄ Lassen Co. 0.36487⁄ 0 1.00000 1.00000 1.00000⁄ 1.00000 1.00000⁄ 1.00000⁄ 1.00000⁄ 1.00000⁄ 1.00000⁄ 1.00000⁄ Riverside Co. 0.23066⁄ 0.61051⁄ 0 0.83161 0.12463 0.02672⁄ 0.16176 1.00000⁄ 1.00000⁄ 1.00000⁄ 1.00000⁄ 1.00000⁄ San Diego Co. 0.12263⁄ 0.57361⁄ 0.03146⁄ 0 0.15175⁄ 0.20475⁄ 0.1719 1.00000⁄ 1.00000⁄ 1.00000⁄ 1.00000⁄ 1.00000⁄ Ventura Co. 0.16923⁄ 0.59623⁄ 0.14061⁄ 0.02415 0 0.04368 0.43157⁄ 1.00000⁄ 1.00000⁄ 1.00000⁄ 1.00000⁄ 1.00000⁄ Walker Pass 0.24645⁄ 0.66571⁄ 0.0179 À0.00285 0.09846⁄ 0 0.32628 1.00000⁄ 1.00000⁄ 1.00000⁄ 0.99999⁄ 1.00000 Yuma Co. 0.22797⁄ 0.49601⁄ 0.03547 0.09833⁄ 0.26087⁄ 0.14057⁄ 0 1.00000⁄ 1.00000⁄ 1.00000⁄ 1.00000⁄ 1.00000⁄ E. nsp Monterey Co. 0.90946⁄ 0.82337⁄ 0.88377⁄ 0.86449⁄ 0.96178⁄ 0.92952⁄ 0.89841⁄ 0 1.00000⁄ 0.55969⁄ 1.00000⁄ 1.00000⁄ Pinnacles NP 0.87278⁄ 0.76801⁄ 0.87805⁄ 0.86189⁄ 0.95293⁄ 0.92643⁄ 0.85269⁄ 0.88040⁄ 0 0.10714⁄ 1.00000⁄ 1.00000⁄ Shell Creek 0.83948⁄ 0.76928⁄ 0.86320⁄ 0.84381⁄ 0.90287⁄ 0.90356⁄ 0.84329⁄ 0.24149⁄ 0.28421 0 1.00000⁄ 1.00000⁄ E. euterpe Carrizo Plain 0.78746⁄ 0.66554⁄ 0.80062⁄ 0.78477⁄ 0.86825⁄ 0.8593⁄ 0.78972⁄ 0.93455⁄ 0.91419⁄ 0.89153⁄ 0 1.00000⁄ Walker Basin 0.80958⁄ 0.64700⁄ 0.83138⁄ 0.81362⁄ 0.90028⁄ 0.88751⁄ 0.79645⁄ 0.94748⁄ 0.91353⁄ 0.88700⁄ 0.72257⁄ 0
Pairwise Ust were computed with Arlequin. Pairwise genetic Jost’s D values were computed in R with R script from Pennings et al. (2011). bold values indicate significant p- values 60.05. P-values are in supplementary materials. D. Rubinoff et al. / Biological Conservation 184 (2015) 278–289 287
Fig. 8. Statistical parsimony network based on COI (1435 bp) data containing 245 Euproserpinus accessions. Circles represent the 76 unique haplotypes and their sizes are proportional to the number of haplotypes. Colors represent different Euproserpinus populations and pie graphs indicate the proportion of each haplotype represented by different color-coded populations. Black dots indicate missing intermediate haplotypes (mutational steps). The combination of unique haplotypes and geographic isolation support the recognition of the three species designated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) region, because it would most likely have experienced the opposite typical mesic-dependent species often studied in the context of temporal patterns of isolation and reconnection. Ice Age speciation events. Further research on insects, particularly xerophilic taxa, may provide additional perspectives on the dynamics of speciation both during, and in between past glaciation 5. Conclusion events. Our study also suggests that more attention be given to insect phylogeography in California. While both E. phaeton and, For Euproserpinus, the Transverse and Southern Sierra ranges to a lesser extent, the new species in the Central Coast appear to and the Central Coast were the major genetic barriers. South of be relatively secure, patterns of speciation and isolation in Eupros- the Transverse Ranges and east of the Sierras is E. phaeton, north erpinus imply the existence of more insect lineages than are cur- is E. euterpe, and then northwest is the new species. Calsbeek rently appreciated, and some may be in need of conservation et al. (2003) and Chatzimanolis and Caterino (2007) review many attention (eg Caterino and Chatzimanolis, 2009). The loss of these studies that also found the Transverse range to be a major barrier species will degrade our ability to understand the phylogeography across multiple classes of animal but, again, the divisions were of one of North America’s most diverse biological provinces. east–west rather than north–south as we found in Euproserpinus. Our study demonstrates that conservation of the Federally list- ed E. euterpe requires a population-level assessment of genetic Author contributions diversity. Our data suggests that E. euterpe from Walker Basin and Carrizo Plain should not be considered a single population, D.R. conceived of the study, wrote the paper, collected samples, because they share no haplotypes in common and have significant and conducted some molecular work. M.S. conducted molecular genetic differentiation (Ust = 0.72 P-value = 0.00; Jost’s D = 0.99 P- work, ran phylogenetic and population genetic analyses and value = 0.00); managing them as separate entities dramatically helped write the paper, P.J. collected samples and assisted with reduces the management areas for the species, particularly the writing, K.O. collected samples and assisted with writing, R.W. col- Walker Basin population, which has lost the vast majority of its lected samples, J. L. R. assisted with molecular work, analysis and habitat to anthropogenic disturbance. Thus, although a new writing. population of E. euterpe has been discovered in the Carrizo Plain, each population represents a unique subset of the genetic diversity Data accessibility of the species. The populations in Walker Basin are imminently threatened by agriculture and housing developments, while DNA sequences: GenBank accessions KP263128-KP263236 populations in the Carrizo Plain suffer habitat degradation from sheep grazing and invasive weeds. Both populations require active Acknowledgements conservation management and protection. The complex pattern of genetic divergence in Euproserpinus Field assistance, specimens or advice was provided by J. Bundy, contributes to a finer understanding of the complex biogeo- J.Smith, L. Crabtree, Peter Jump, P. Opler and D. Bowman. Permits graphical history of California. Additional markers which are infor- and organizational support were provided by U.S. Fish and Wildlife mative at the species/population level may provide important Service, NPS and BLM. We thank the Kern Primrose Sphinx moth additional resolution regarding the relationships within the genus. working group, organized by K. Sharum, for long term support Euproserpinus demonstrates the importance of studying taxa and advice. Funding was provided via G. Hinshaw, V. Bloom, and whose response to glaciation is likely the inverse of the more D. Kelly (USFWS). Additional support was provided by A. Kuritsubo 288 D. Rubinoff et al. / Biological Conservation 184 (2015) 278–289 and K. Sharum (BLM) in conjunction with T. Longcore (Urban Wild- Fu, Y.X., Li, W.H., 1993. Statistical tests of neutrality of mutations. Genetics 133, lands group), via agreement L09AC15868-0003-0000 with The 693–709. Gompert, Z., Nice, C.C., Fordyce, J.A., Forister, M.L., Shapiro, A.M., 2006. Identifying Urban Wildlands Group, and the National Park Service (PINN- units for conservation using molecular systematics: the cautionary tale of the 131046) for DNA analysis of specimens from Pinnacles National Karner blue butterfly. Molecular Ecol. 15, 1759–1768. Park. 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